Disintegrin variants and their use in treating osteoporosis-induced bone loss and angiogenesis-related diseases

Abstract

Disintegrin variants and pharmaceutical uses thereof are disclosed. The disintegrin variant includes an isolated polypeptide that has integrin αvβ3 receptor-antagonist activity and substantially reduced integrin αllbβ3 and/or α5β1 receptor-blocking activity as compared to a wild-type disintegrin. The variant is encoded by a modified disintegrin nucleotide sequence that encodes a modified amino acid sequence, resulting in a polypeptide having substantially reduced affinity to integrin αllbβ3 and/or α5β1 as compared to a wild-type disintegrin. The variant is useful for treatment and/or prevention of αvβ3 integrin-associated diseases in a mammal, which include osteoporosis, bone tumor or cancer growth, angiogenesis-related tumor growth and metastasis, tumor metastasis in bone, malignancy-induced hypercalcemia, angiogenesis-related eye diseases, Paget's disease, rheumatic arthritis, and osteoarthritis. The angiogenesis-related eye diseases include age-related macular degeneration, diabetic retinopathy, corneal neovascularizing diseases, ischaemia-induced neovascularizing retinopathy, high myopia, and retinopathy of prematurity.

1. An isolated polypeptide selective for αvβ3 integrin, wherein the polypeptide is a variant of a disintegrin which comprises a variant of SEQ ID NO: 29 that contains an amino acid substitution at position 3 and an optional substitution at one or more positions 1, 5 and 6 of SEQ ID NO: 29, or a pharmaceutically acceptable salt of said isolated polypeptide. 
     
     
       2. The isolated polypeptide of  claim 1 , wherein the disintegrin is selected from the group consisting of rhodostomin, albolabrin, applagin, basilicin, batroxostatin, bitistatin, cereberin, cerastin, crotatroxin, durissin, elegantin, flavoridin, flavostatin, halysin, halystatin, jararacin, jarastatin, kistrin, lachesin, lutosin, molossin, salmosin, saxatilin, tergeminin, trimestatin, trimucrin, trimutase, ussuristatin, and viridin. 
     
     
       3. The isolated polypeptide of  claim 1 , wherein the disintegrin comprises a variant of rhodostomin. 
     
     
       4. The isolated polypeptide of  claim 1 , wherein said isolated polypeptide comprises a variant of SEQ ID NO: 1. 
     
     
       5. The isolated polypeptide of  claim 1 , wherein said isolated polypeptide comprises an amino acid sequence chosen from SEQ ID NOs: 36-40. 
     
     
       6. The isolated polypeptide of  claim 1 , wherein the amino acid substitution at position 3 is chosen from Leu, lle and His and the optional amino acid substitutions are chosen from Ala at position 1, Asp, Met, and Asn at position 5, and Val, Leu, and Met at position 6. 
     
     
       7. The isolated polypeptide of  claim 1 , wherein the variant of SEQ ID NO: 29 contains an amino acid substitution at position 5. 
     
     
       8. The isolated polypeptide of  claim 1 , wherein the variant of SEQ ID NO: 29 contains an amino acid substitution at a position chosen from positions 1, 5 and 6 of SEQ ID NO: 29. 
     
     
       9. The isolated polypeptide of  claim 1 , wherein the variant of SEQ ID NO: 29 contains said amino acid substitutions in at least two positions chosen from positions 1, 5 and 6 of SEQ ID NO: 29. 
     
     
       10. The isolated polypeptide of  claim 9 , wherein the amino acid substitutions are at positions 5 and 6 of SEQ ID NO: 29. 
     
     
       11. The isolated polypeptide of  claim 1 , wherein the variant of SEQ ID NO: 29 contains said amino acid substitutions at positions 1, 5 and 6 of SEQ ID NO: 29. 
     
     
       12. The isolated polypeptide of  claim 1 , wherein said isolated polypeptide is encoded by a polynucleotide comprising a nucleotide sequence chosen from SEQ ID NOs: 50-54. 
     
     
       13. The isolated polypeptide of  claim 1 , wherein said isolated polypeptide exhibits at least about a 5-fold decrease in affinity to allbβ3 and/or a5β1 as compared to wild-type rhodostomin. 
     
     
       14. The isolated polypeptide of  claim 1 , wherein said isolated polypeptide exhibits at least about a 200-fold decrease in affinity to allbβ3 and/or a5β1 as compared to wild-type rhodostomin. 
     
     
       15. The isolated polypeptide of  claim 1 , wherein said isolated polypeptide exhibits at least about a 1000-fold decrease in affinity to allbβ3 and/or a5β1 as compared to wild-type rhodostomin. 
     
     
       16. The isolated polypeptide of  claim 1 , wherein said isolated polypeptide exhibits at least about 5-fold decrease in ability to aggregate platelets as compared to wild-type rhodostomin. 
     
     
       17. The isolated polypeptide of  claim 1 , wherein said isolated polypeptide exhibits reduced activity in prolongation of blood clotting time as compared to wild-type rhodostomin. 
     
     
       18. The isolated polypeptide of  claim 1 , wherein said isolated polypeptide is conjugated with albumin or pegylated. 
     
     
       19. A physiologically acceptable composition comprising the isolated polypeptide of  claim 1 , or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 
     
     
       20. A physiologically acceptable composition comprising the isolated polypeptide of  claim 8 , or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 
     
     
       21. A physiologically acceptable composition comprising the isolated polypeptide of  claim 9 , or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 
     
     
       22. A physiologically acceptable composition comprising the isolated polypeptide of  claim 11 , or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 
     
     
       23. A physiologically acceptable composition comprising the isolated polypeptide of  claim 12 , or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 
     
     
       24. The physiologically acceptable composition of  claim 19 , wherein said isolated polypeptide is conjugated with albumin or pegylated. 
     
     
       25. An isolated polypeptide comprising an amino acid sequence chosen from SEQ ID NOs: 63-67, or a pharmaceutically acceptable salt of said isolated polypeptide. 
     
     
       26. A method for making the isolated polypeptide according to  claim 1 , comprising the steps of:
 a. transfecting a host cell with a polynucleotide encoding said polypeptide; 
 b. growing said host cell in a culture medium; and 
 c. isolating said polypeptide. 
 
     
     
       27. The method of  claim 26 , wherein (b) further comprises adding methanol to said culture medium. 
     
     
       28. The method of  claim 26 , further comprising growing the host cell in a culture medium free of amino acids. 
     
     
       29. The method of  claim 26 , wherein (c) further comprises performing a column chromatography to obtain said polypeptide. 
     
     
       30. The method of  claim 26 , further comprising performing a HPLC to obtain said polypeptide. 
     
     
       31. The method of  claim 26 , wherein said host cell is a yeast cell. 
     
     
       32. An isolated polypeptide selective for avβ3 integrin encoded by a polynucleotide that hybridizes to the polynucleotide encoding a polypeptide selective for avβ3 integrin wherein said isolated polypeptide comprises a variant of SEQ ID NO: 29 that contains an amino acid substitution at position 3 and an optional substitution at one or more positions 1, 5 and 6 of SEQ ID NO: 29, or a pharmaceutically acceptable salt of said isolated polypeptide. 
     
     
       33. An isolated polypeptide comprising SEQ ID NO: 38, or a pharmaceutically acceptable salt of said isolated polypeptide. 
     
     
       34. An isolated polypeptide comprising SEQ ID NO: 65, or a pharmaceutically acceptable salt of said isolated polypeptide. 
     
     
       35. An isolated polypeptide wherein said isolated polypeptide is encoded by a polynucleotide comprising SEQ ID NO: 52, or a pharmaceutically acceptable salt of said isolated polypeptide. 
     
     
       36. The isolated polypeptide of  claim 33 , wherein said isolated polypeptide exhibits at least about a 2000-fold decrease in affinity to αllbβ3 as compared to wild-type rhodostomin. 
     
     
       37. The isolated polypeptide of  claim 33 , wherein said isolated polypeptide exhibits at least about a 90-fold decrease in affinity to α5β1 as compared to wild-type rhodostomin. 
     
     
       38. The isolated polypeptide of  claim 34 , wherein said isolated polypeptide exhibits at least about a 2000-fold decrease in affinity to αllbβ3 as compared to wild-type rhodostomin. 
     
     
       39. The isolated polypeptide of  claim 34 , wherein said isolated polypeptide exhibits at least about a 90-fold decrease in affinity to αllbβ1 as compared to wild-type rhodostomin. 
     
     
       40. The isolated polypeptide of  claim 33 , wherein said isolated polypeptide is conjugated with albumin or pegylated. 
     
     
       41. The isolated polypeptide of  claim 34 , wherein said isolated polypeptide is conjugated with albumin or pegylated. 
     
     
       42. The isolated polypeptide of  claim 35 , wherein said isolated polypeptide is conjugated with albumin or pegylated. 
     
     
       43. The isolated polypeptide of  claim 36 , wherein said isolated polypeptide is conjugated with albumin or pegylated. 
     
     
       44. The isolated polypeptide of  claim 37 , wherein said isolated polypeptide is conjugated with albumin or pegylated. 
     
     
       45. The isolated polypeptide of  claim 38 , wherein said isolated polypeptide is conjugated with albumin or pegylated. 
     
     
       46. The isolated polypeptide of  claim 39 , wherein said isolated polypeptide is conjugated with albumin or pegylated. 
     
     
       47. A physiologically acceptable composition comprising the isolated polypeptide of  claim 33 , or a pharmaceutically acceptable salt of said isolated polypeptide, and a pharmaceutically acceptable carrier. 
     
     
       48. A physiologically acceptable composition comprising the isolated polypeptide of  claim 34 , or a pharmaceutically acceptable salt of said isolated polypeptide, and a pharmaceutically acceptable carrier. 
     
     
       49. A physiologically acceptable composition comprising the isolated polypeptide of  claim 35 , or a pharmaceutically acceptable salt of said isolated polypeptide, and a pharmaceutically acceptable carrier. 
     
     
       50. A physiologically acceptable composition comprising the isolated polypeptide of  claim 36 , or a pharmaceutically acceptable salt of said isolated polypeptide, and a pharmaceutically acceptable carrier. 
     
     
       51. A physiologically acceptable composition comprising the isolated polypeptide of  claim 37 , or a pharmaceutically acceptable salt of said isolated polypeptide, and a pharmaceutically acceptable carrier. 
     
     
       52. A physiologically acceptable composition comprising the isolated polypeptide of  claim 38 , or a pharmaceutically acceptable salt of said isolated polypeptide, and a pharmaceutically acceptable carrier. 
     
     
       53. A physiologically acceptable composition comprising the isolated polypeptide of  claim 39 , or a pharmaceutically acceptable salt of said isolated polypeptide, and a pharmaceutically acceptable carrier. 
     
     
       54. A physiologically acceptable composition comprising the isolated polypeptide of  claim 40 , or a pharmaceutically acceptable salt of said isolated polypeptide, and a pharmaceutically acceptable carrier. 
     
     
       55. A physiologically acceptable composition comprising the isolated polypeptide of  claim 41 , or a pharmaceutically acceptable salt of said isolated polypeptide, and a pharmaceutically acceptable carrier. 
     
     
       56. A physiologically acceptable composition comprising the isolated polypeptide of  claim 42 , or a pharmaceutically acceptable salt of said isolated polypeptide, and a pharmaceutically acceptable carrier. 
     
     
       57. A physiologically acceptable composition comprising the isolated polypeptide of  claim 43 , or a pharmaceutically acceptable salt of said isolated polypeptide, and a pharmaceutically acceptable carrier. 
     
     
       58. A physiologically acceptable composition comprising the isolated polypeptide of  claim 44 , or a pharmaceutically acceptable salt of said isolated polypeptide, and a pharmaceutically acceptable carrier. 
     
     
       59. A physiologically acceptable composition comprising the isolated polypeptide of  claim 45 , or a pharmaceutically acceptable salt of said isolated polypeptide, and a pharmaceutically acceptable carrier. 
     
     
       60. A physiologically acceptable composition comprising the isolated polypeptide of  claim 46 , or a pharmaceutically acceptable salt of said isolated polypeptide, and a pharmaceutically acceptable carrier. 
     
     
       61. A method for treatment of a disease selected from the group consisting of osteoporosis-induced bone loss and an angiogenesis-related disease comprising:
 administering to a mammal in need thereof a therapeutically effective amount of the isolated polypeptide of  claim 1 , or a pharmaceutically acceptable salt thereof. 
 
     
     
       62. The method of  claim 61 , wherein said isolated polypeptide comprises an amino acid sequence chosen from SEQ ID NOs: 36-40. 
     
     
       63. The method of  claim 61 , wherein said isolated polypeptide comprises an amino acid sequence chosen from SEQ ID NOs: 63-67. 
     
     
       64. The method of  claim 61 , wherein said isolated polypeptide is pegylated or conjugated with albumin. 
     
     
       65. The method of  claim 61 , wherein the angiogenesis-related disease is chosen from angiogenesis-related tumor growth and metastasis and an angiogenesis-related eye disease. 
     
     
       66. The method of  claim 65 , wherein the angiogenesis-related eye disease is chosen from age-related macular degeneration, diabetic retinopathy, corneal neovascularizing diseases, ischaemia-induced neovascularizing retinopathy, high myopia, and retinopathy of prematurity. 
     
     
       67. The method of  claim 61 , wherein the mammal is a human. 
     
     
       68. A method for inhibition of osteoclasts and/or angiogenesis comprising:
 administering to a mammal in need thereof a therapeutically effective amount of the isolated polypeptide of  claim 1 , or a pharmaceutically acceptable salt thereof. 
 
     
     
       69. The method of  claim 68 , wherein said isolated polypeptide comprises amino acid sequence chosen from SEQ ID NOs: 36-40. 
     
     
       70. The method of  claim 68  wherein said isolated polypeptide comprises an amino acid sequence chosen from SEQ ID NOs: 63-67. 
     
     
       71. The method of  claim 68 , wherein said isolated polypeptide is pegylated or conjugated with albumin. 
     
     
       72. The method of  claim 68 , wherein the mammal is a human.